We intend to continue our study of mechanisms of enzyme reactions where the stereochemistry is cryptic due to these reactions taking place at pro-chiral centers. Our goals are to study L-threonine dehydrogenase, aminoacetone synthase, DL-1-amino-2-propanol dehydrogenase, and DL-aminopropanol-OP phospholyase. These enzymes are all involved in the metabolism of the essential amino acid L-threonine and one of its key metabolites, aminoacetone. Significant amounts of aminoacetone are produced in the liver, and metabolic anomalies have been identified in cases of liver disease and of a number of sarcomas and carcinomas. Two of the enzymes to be studied utilize pyridoxal phosphate as coenzyme, and two use NAD. With each enzyme reaction we will prepare and use substrates chirally labeled with either deuterium or tritium, and analyze the labeling of the enzymatic products to reveal the stereochemical course of the reaction. We will also trap tritium labeled enzyme-coenzyme adducts and substrate-coenzyme adducts by in situ reduction with (3H)-sodium borohydride. These adducts will be isolated and the chirality of the labeled centers analyzed to determine whether significant conformational changes have occurred upon binding of the substrates. A recurrent and very important feature of the work proposed is the use of 3H NMR as a routine analytical tool where the very small masses of product obtainable prevent the use of deuterium labels and the more traditional 1H and 2H NMR spectroscopy. If successful, our approach will save substantial amounts of time over the currently accepted approach of extensive chemical degradations to configurational standards followed by enzymatic analyses.